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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

All the three required in vitro genotoxicity studies were performed on 3,3,5 -trimethylcyclohexyl acrylate. Positive results were observed in the In vitro gene mutation study in bacteria (OECD 471), however negative results were obtained in the In vitro cytogenicity / micronucleus study (OECD 487) and in the In vitro gene mutation study in mammalian cells (OECD 476). Based on the weight of evidence, 3,3,5 -trimethylcyclohexyl acrylate is considered to be not mutagenic.

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Endpoint:
in vitro cytogenicity / micronucleus study
Type of information:
experimental study
Adequacy of study:
key study
Study period:
09 May 2016 - 22 June 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
Version / remarks:
26 September 2014
Deviations:
no
Principles of method if other than guideline:
The historical data used for the validation of long treatment period without S9 mix (24 hours treatment + 0 hour recovery) were generated with non-audited data from non-GLP studies. These data were performed in compliance with CiToxLAB France’s standard operating procedures. Since CiToxLAB France is a Test facility certified by the French National Authorities for Good Laboratory Practice, and the procedures undertaken are the same, this deviation is considered not to prejudice the overall GLP status of the study and the scientific reliability of the study conclusions. Moreover, the corresponding mean frequency of micronucleated cells in the vehicle control was 2‰ in this experiment, therefore = 5‰ as specified in the acceptance criteria.
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian cell micronucleus test
Target gene:
Not applicable (not a gene mutation assay).
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Type and identity of media: RPMI 1640 medium containing 10% heat inactivated horse serum, L-Glutamine (2 mM), penicillin (100 U/mL), streptomycin (100 µg/mL) and sodium pyruvate (200 µg/mL)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes

L5178Y TK+/- cells are an established cell line recommended by international regulations for in vitro mammalian cell gene mutation test and for in vitro micronucleus test. Indeed, they are suitable to reveal chemically induced micronuclei. The average cell cycle time is approximately 10-12 hours.
L5178Y TK+/- cells were obtained from ATCC (American Type Culture Collection, Manassas, USA), by the intermediate of Biovalley (Marne-La-Vallée, France).
The cells were stored in a cryoprotective medium (10% horse serum and 10% dimethylsulfoxide (DMSO)) at -80°C and each batch of frozen cells was checked for the absence of mycoplasma.

Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
rat liver S9 mix
Test concentrations with justification for top dose:
Since the test item was found cytotoxic and poorly soluble in the culture medium during the preliminary test, the selection of the highest dose-level to be used in the main experiments was based on the level of cytotoxicity and/or on the level of precipitate, according to the criteria specified in the international guidelines.

Experiment without S9 mix
With a treatment volume of 0.5% (v/v) in culture medium, the dose-levels selected were 0.000625, 0.00125, 0.0025, 0.005, 0.01, 0.015, 0.02 and 0.04 mM, for the 3- and 24-hour treatments.

Experiments with S9 mix
With a treatment volume of 0.5% (v/v) in culture medium, the selected dose-levels were as follows:
- 0.125, 0.25, 0.5, 1, 2 and 4 mM for the first experiment,
- 0.0009, 0.003, 0.008, 0.025, 0.074, 0.222, 0.667 and 2 mM for the second experiment.
Vehicle / solvent:
- Vehicle used: dimethylsulfoxide (DMSO)
- Justification for choice: Using a test item concentration of 390 mg/mL in the vehicle (DMSO) and a treatment volume of 0.5% (v/v) in culture medium, the highest recommended dose-level of 10 mM (corresponding to 1950 µg/mL) was achievable.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: mitomycin C, colchicine (-S9 mix); cyclophosphamide (+S9 mix)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
Preliminary cytotoxicity test
Without S9 mix 3 h treatment + 24 h recovery
24 h treatment + 0 h recovery
With S9 mix 3 h treatment + 24 h recovery

First cytogenetic experiment
Without S9 mix 3 h treatment + 24 h recovery
24 h treatment + 0 h recovery
With S9 mix 3 h treatment + 24 h recovery

Second cytogenetic experiment
With S9 mix 3 h treatment + 24 h recovery

NUMBER OF CELLS EVALUATED: 2000 mononucleated cells / dose

DETERMINATION OF CYTOTOXICITY
- Method: population doubling
Evaluation criteria:
The biological relevance of the results was always taken into account when evaluating results.

Evaluation of a positive response: a test item is considered to have clastogenic and/or aneugenic potential, if all the following criteria were met:
- a dose-related increase in the frequency of micronucleated cells was demonstrated by a statistically significant trend test,
- for at least one dose-level, the frequency of micronucleated cells of each replicate culture was above the corresponding vehicle historical range,
- a statistically significant difference in comparison to the corresponding vehicle control was obtained at one or more dose-levels.

Evaluation of a negative response: a test item is considered clearly negative if none of the criteria for a positive response was met.
Statistics:
yes
Key result
Species / strain:
other: mouse lymphoma L5178Y TK+/- cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: none
- Effects of osmolality: none
- Evaporation from medium: none
- Emulsion: in both experiments with S9 mix, an emulsion was observed in the culture medium at the end of the treatment period, at dose-levels >= 2 mM.
- Definition of acceptable cells for analysis: Analysis was performed under a microscope (1000 x magnification), on the basis of the recommendations of Miller et al. (1995), according to the following criteria:
* micronuclei should be clearly surrounded by a nuclear membrane,
* the micronucleus area should be less than one-third of the area of the main nucleus,
* non-refractility of the micronuclei,
* micronuclei should not be linked to the main nucleus via nucleoplasmic bridges,
* micronuclei should be located within the cytoplasma of the cell,
* only mononucleated cells with a number of micronuclei <= 5 should be scored to exclude apoptosis and nuclear fragmentation.

- Other confounding effects: none.

RANGE-FINDING/SCREENING STUDIES:
Using a test item concentration of 390 mg/mL in the vehicle and a treatment volume of 0.5% (v/v) in culture medium, the highest recommended dose-level of 10 mM (corresponding to 1950 µg/mL) was achievable. Thus, the dose-levels selected for the treatment of the preliminary test were 0.0002, 0.002, 0.02, 0.2, 1, 2, 5 and 10 mM.

At the highest dose-level of 10 mM, the pH of the culture medium was approximately 7.4 (as for the vehicle control) and the osmolality was 355 mOsm/kg H2O (379 mOsm/kg for the vehicle control). Therefore, none of the selected dose-levels was considered to produce extreme culture conditions and the highest recommended dose-level of 10 mM could be selected as the highest dose-level for the main experiment.

An emulsion was observed in the culture medium at dose-levels >= 2 mM at the end of the 3-h treatment period and at dose-levels >= 5 mM at the end of the 24-h treatment period.

Following the 3- and 24-hour treatments without S9 mix, a marked cytotoxicity was observed at dose levels >= 0.02 mM, as shown by a 60 to 100% decrease in the PD.
Following the 3-hour treatment with S9 mix, a 33 to 55% decrease in the PD was observed, without any clear evidence of a dose-response relationship, at the dose-levels of 0.2, 2 and 10 mM.

MAIN STUDY
Experiment without S9 mix
With a treatment volume of 0.5% (v/v) in culture medium, the dose-levels selected were 0.000625, 0.00125, 0.0025, 0.005, 0.01, 0.015, 0.02 and 0.04 mM, for the 3- and 24-hour treatments.
 
No emulsion was observed in the culture medium at any of the tested dose-levels, either at the beginning or the end of the treatment periods.

Following the 3- and 24-hour treatments, a slight to severe cytotoxicity was induced at dose-levels = 0.01 mM, as shown by a 26 to 100% decrease in the PD.

The dose-levels selected for micronucleus analysis were 0.005, 0.01 and 0.015 mM for the 3- and 24-hour treatments,the latter inducing a 45 and 26% decrease in the PD, respectively, and higher dose-levels being too cytotoxic.
It is to be noted that the highest analyzable dose-level of 0.015 mM did not exhibit about 55% cytotoxicity for either treatment period. Considering the narrow dose-levels spacing used, the overall available results were considered to be suitable to allow a reliable interpretation.
 
Following the 3- and 24-hour treatments, neither statistically significant nor dose-related increase in the frequency of micronucleated cells was noted at any of the tested dose-levels relative to the vehicle controls. None of the analyzed dose-levels showed frequency of micronucleated cells of each replicate culture above the vehicle control historical range.
 
These results met the criteria of a negative response.
 
Experiments with S9 mix
With a treatment volume of 0.5% (v/v) in culture medium, the selected dose-levels were as follows:
- 0.125, 0.25, 0.5, 1, 2 and 4 mM for the first experiment,
- 0.0009, 0.003, 0.008, 0.025, 0.074, 0.222, 0.667 and 2 mM for the second experiment.
 
In both experiments, an emulsion was observed in the culture medium at the end of the treatment period, at dose-levels = 2 mM.

In the first experiment, a moderate to severe cytotoxicity was induced from the lowest tested dose-level, as shown by a 48 to 100% decrease in the PD. Since the cytotoxicity obtained in this experiment was higher than expected (based on the results of the preliminary test), not enough analyzable dose-levels were available for the analysis of micronuclei. 
In the second experiment, performed under the same experimental conditions, but using a lower and wider range of dose-levels, no cytotoxicity was observed at any of the tested dose-levels, as shown by the absence of any noteworthy decrease in the PD (i.e. results consistent with those of the preliminary test).

The dose-levels selected for micronucleus analysis (second experiment) were 0.222, 0.667 and 2 mM, the latter being the lowest dose-level showing test item emulsion in the culture medium at the end of the treatment period.
 
Neither statistically significant nor dose-related increase in the frequency of micronucleated cells was noted at any of the tested dose-levels relative to the vehicle control. None of the analyzed dose-levels showed frequency of micronucleated cells of each replicate culture above the vehicle control historical range.
 
These results met the criteria of a negative response.
Conclusions:
3.3.5-trimethylcyclohexyl acrylate did not induce any chromosome damage, or damage to the cell division apparatus, in cultured mammalian somatic cells, using L5178Y TK+/-mouse lymphoma cells, either in the presence or absence of a rat liver metabolizing system.
 
Executive summary:

The objective of this study was to evaluate the potential of the test item, to induce an increase in the frequency of micronucleated cellsin the mouse cell line L5178Y TK+/-.

 

After a preliminary cytotoxicity test, the test item, diluted in dimethylsulfoxide (DMSO), was tested in a first experiment, with (3h of treatment) and without (3 h and 24h of treament) a metabolic activation system, the S9 mix, prepared from a liver microsomal fraction (S9 fraction) of rats induced with Aroclor 1254.

 

Since not enough analyzable dose-levels were obtained following the treatment of the first experiment with S9 mix, the corresponding slide analysis was not performed and the test item was tested in a second experiment with S9 mix using the same conditions (3 h treatment + 24 h recovery).

 

Each treatment was coupled to an assessment of cytotoxicity at the same dose-levels. Cytotoxicity was evaluated by determining the PD (Population Doubling) of cells.

Then, after the final cell counting, the cells were washed and fixed. Then, cells from three dose-levels of the test item-treated cultures were dropped onto clean glass slides (except for the first experiment with S9 mix). The slides were air-dried before being stained in 5% Giemsa. Slides from vehicle and positive controls cultures were also prepared as described above. All slides were coded before analysis, so that the analyst was unaware of the treatment details of the slide under evaluation ("blind" scoring). For the first experiment without S9 mix and the second experiment with S9 mix, micronuclei were analyzed for three dose-levels of the test item, for the vehicle and the positive controls, in 1000 mononucleated cells per culture (total of 2000 mononucleated cells per dose).

Number of cells with micronuclei and number of micronuclei per cell were recorded separately for each treated and control culture.

 

Since the test item was found cytotoxic and poorly soluble in the culture medium during the preliminary test, the selection of the highest dose-level to be used in the main experiments was based on the level of cytotoxicity and/or on the level of precipitate, according to the criteria specified in the international guidelines.

The mean population doubling and the mean frequencies of micronucleated cells for the vehicle controls were as specified in the acceptance criteria. Also, positive control cultures showed clear statistically significant increases in the frequency of micronucleated cells. The study was therefore considered to be valid.

 

Experiment without S9 mix

With a treatment volume of 0.5% (v/v) in culture medium, the dose-levels selected were 0.000625, 0.00125, 0.0025, 0.005, 0.01, 0.015, 0.02 and 0.04 mM, for the 3- and 24-hour treatments.

 

No emulsion was observed in the culture medium at any of the tested dose-levels, either at the beginning or the end of the treatment periods.


Following the 3- and 24-hour treatments, a slight to severe cytotoxicity was induced at dose-levels = 0.01 mM, as shown by a 26 to 100% decrease in the PD.

The dose-levels selected for micronucleus analysis were 0.005, 0.01 and 0.015 mM for the 3- and 24-hour treatments,the latter inducing a 45 and 26% decrease in the PD, respectively, and higher dose-levels being too cytotoxic.

It is to be noted that the highest analyzable dose-level of 0.015 mM did not exhibit about 55% cytotoxicity for either treatment period. Considering the narrow dose-levels spacing used, the overall available results were considered to be suitable to allow a reliable interpretation.

 

Following the 3- and 24-hour treatments, neither statistically significant nor dose-related increase in the frequency of micronucleated cells was noted at any of the tested dose-levels relative to the vehicle controls. None of the analyzed dose-levels showed frequency of micronucleated cells of each replicate culture above the vehicle control historical range.

 

These results met the criteria of a negative response.

 

Experiments with S9 mix

With a treatment volume of 0.5% (v/v) in culture medium, the selected dose-levels were as follows:

- 0.125, 0.25, 0.5, 1, 2 and 4 mM for the first experiment,

- 0.0009, 0.003, 0.008, 0.025, 0.074, 0.222, 0.667 and 2 mM for the second experiment.

 

In both experiments, an emulsion was observed in the culture medium at the end of the treatment period, at dose-levels = 2 mM.

In the first experiment, a moderate to severe cytotoxicity was induced from the lowest tested dose-level, as shown by a 48 to 100% decrease in the PD. Since the cytotoxicity obtained in this experiment was higher than expected (based on the results of the preliminary test), not enough analyzable dose-levels were available for the analysis of micronuclei. 

In the second experiment, performed under the same experimental conditions, but using a lower and wider range of dose-levels, no cytotoxicity was observed at any of the tested dose-levels, as shown by the absence of any noteworthy decrease in the PD (i.e. results consistent with those of the preliminary test).

The dose-levels selected for micronucleus analysis (second experiment) were 0.222, 0.667 and 2 mM, the latter being the lowest dose-level showing test item emulsion in the culture medium at the end of the treatment period.

 

Neither statistically significant nor dose-related increase in the frequency of micronucleated cells was noted at any of the tested dose-levels relative to the vehicle control. None of the analyzed dose-levels showed frequency of micronucleated cells of each replicate culture above the vehicle control historical range.

 

These results met the criteria of a negative response.

Under the experimental conditions of the study, 3.3.5-trimethylcyclohexyl acrylate, the test item did not induce any chromosome damage, or damage to the cell division apparatus, in cultured mammalian somatic cells, using L5178Y TK+/- mouse lymphoma cells, either in the presence or absence of a rat liver metabolizing system.

 

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
30 November 2015 -- 08 February 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidine operon
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
S. typhimurium TA 102
Metabolic activation:
with and without
Metabolic activation system:
rat liver S9 mix
Test concentrations with justification for top dose:
Experiments without S9 mix
Selected dose-levels ranged from 1.3 to 5000 µg/plate.

Experiments with S9 mix
Selected dose-levels ranged from 39.1 to 5000 µg/plate.

Vehicle / solvent:
- Vehicle used: dimethylsulfoxide (DMSO)
- Justification for choice: using a test item concentration of 100 mg/mL in the vehicle and a treatment volume of 50 µL/plate, the highest recommended dose-level of 5000 µg/plate was achievable. Thus, the dose-levels selected for the preliminary test were 10, 100, 500, 1000, 2500 and 5000 µg/plate.

Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: sodium azide, 9-aminoacridine, 2-nitrofluorene, mitomycin C (-S9 mix); 2-anthramine, benzo(a)pyrene (+S9 mix)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar

DURATION
- Preincubation period: 60 minutes
- Exposure duration: 48 to 72 hours.

DETERMINATION OF CYTOTOXICITY
- Method: decrease in number of revertant colonies and/or thinning of the bacterial lawn
Evaluation criteria:
A reproducible 2-fold increase (for the TA 98, TA 100 and TA 102 strains) or 3-fold increase (for the TA 1535 and TA 1537 strains) in the number of revertants compared with the vehicle controls, in any strain at any dose-level and/or evidence of a dose-relationship was considered as a positive result. Reference to historical data, or other considerations of biological relevance may also be taken into account.
Statistics:
no
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium, other: TA1537, TA98, TA100, TA102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
The mean number of revertants for the vehicle and positive controls met the acceptance criteria. Also, there were at least five analysable dose-levels for each strain and test condition. The study was therefore considered to be valid.

Experiments without S9 mix
The selected dose-levels were:
. 312.5, 625, 1250, 2500 and 5000 µg/plate for the TA 102 strain in both mutagenicity experiments and for the TA 98 strain in the first experiment,
. 156.3, 312.5, 625, 1250, 2500 and 5000 µg/plate for the TA 98 strain in the second experiment,
. 39.1, 78.1, 156.3, 312.5, 625 and 1250 µg/plate for the TA 100 strain in the first experiment,
. 19.5, 39.1, 78.1, 156.3, 312.5 and 625 µg/plate for the TA 1535 strain in both experiments and for the TA 100 strain in the second experiment,
. 1.3, 3.9, 11.6, 34.7, 104.2 and 312.5 µg/plate for the TA 1537 strain in the first experiment,
. 4.9, 9.8, 19.5, 39.1, 78.1 and 156.3 µg/plate for the TA 1537 strain in the second experiment.
No precipitate was observed in the Petri plates when scoring the revertants at any of the tested dose-levels.
A moderate to strong toxicity was noted at dose-levels = 104.2 µg/plate in the TA 1537 strain, = 156.3 µg/plate in the TA 100 strain, = 312.5 µg/plate in the TA 1535 strain and = 1250 µg/plate in the TA 98 strain. No noteworthy toxicity was noted at any dose-levels in the TA 102 strain.
The test item did not induce any noteworthy increase in the number of revertants, in any of the five tested strains, in either experiment.
These results without S9 mix met the criteria of a negative response.

Experiments with S9 mix
The selected dose-levels were:
. 312.5, 625, 1250, 2500 and 5000 µg/plate for the five stains in the first experiment,
. 156.3, 312.5, 625, 1250, 2500 and 5000 µg/plate for the TA 102 strain in the second experiment,
. 39.1, 78.1, 156.3, 312.5, 625 and 1250 µg/plate for the TA 1535, TA 1537, TA 98 and TA 100 strains in the second experiment,
. 312.5, 625, 1250, 2500, 3750 and 5000 µg/plate for the TA 1535 strain in the third experiment.
No precipitate was observed in the Petri plates when scoring the revertants at any of the tested dose-levels.
Using the direct plate incorporation method (i.e. in the first and third experiments), a moderate toxicity was only observed at dose-levels = 2500 µg/plate in the TA 1537 strain. No toxicity was observed in the other tested strains.
Using the pre-incubation method (i.e. in the second experiment), a moderate to strong toxicity was noted at dose-levels = 625 µg/plate in the TA 1535, TA 1537, TA 98 and TA 100 strains, and = 1250 µg/plate in the TA 102 strain.
The test item did not induce any noteworthy increase in the number of revertants in the TA 1537, TA 98, TA 100 and TA 102 strains in any experiments, or in the TA 1535 strain in the second experiment performed according to the pre-incubation method.
An increase in the number of revertants was noted at 5000 µg/plate in the TA 1535 strain in the first experiment (direct plate incorporation method). This increase exceeded the positive threshold of 3-fold the vehicle control value (3.6-fold) with an evidence of a dose-response relationship. Moreover, the corresponding mean number of revertants was above the vehicle control historical range (36.3 revertants/plate versus [9-26] for the historical data) as well as two out of three individual values at the lower tested dose level of 2500 µg/plate (22, 30, 31 revertants/plate).
Noteworthy increases in the number of revertants were then observed in the third experiment (under the same experimental conditions, i.e. direct plate incorporation method, and using a narrower range of dose-levels). These increases did not reach the positive threshold of 3-fold the vehicle control value (up to 2.9-fold), however there was an evidence of a dose-response relationship and the mean numbers of revertants at the four higher dose-levels (i.e. = 1250 µg/plate) were above the historical data range (mean numbers of revertants up to 46.3 (50, 40, 49) revertants/plate and individual values up to 53 revertants/plate versus [9-26] for the historical data). Despite the positive threshold was not reached in the confirmatory experiment, the dose-related increases exceeding the historical data were reproducible, and they were thus considered to be biologically relevant.
As a consequence, the results with S9 mix met the criteria of a positive response.
Conclusions:
Under the experimental conditions of this study, the test item showed a mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium TA 1535 strain in the presence of a rat liver metabolizing system, following the direct place incorporation method only.
Executive summary:

The objective of this study was to evaluate the potential of the test item to induce reverse mutations in Salmonella typhimurium.

A preliminary toxicity test was performed to define the dose-levels of 3.3.5-trimethylcyclohexyl acrylate, dissolved in dimethylsulfoxide (DMSO), to be used for the mutagenicity experiments. The test item was then tested in three independent experiments, with or without a metabolic activation system, the S9 mix, prepared from a liver post-mitochondrial fraction (S9 fraction) of rats induced with Aroclor 1254.

Treatments were performed according to the direct plate incorporation method except for the second experiment with S9 mix, which was performed according to the pre-incubation method (60 minutes, 37°C). Five strains of bacteria Salmonella typhimurium were used: TA 1535, TA 1537, TA 98, TA 100 and TA 102. Each strain was exposed to at least five dose-levels of the test item (three plates/dose-level). After 48 to 72 hours of incubation at 37°C, the revertant colonies were scored.

The evaluation of the toxicity was performed on the basis of the observation of the decrease in the number of revertant colonies and/or a thinning of the bacterial lawn.

 

Experiments without S9 mix

A moderate to strong toxicity was noted at dose-levels superior or equal to 104.2 µg/plate in the TA 1537 strain, superior or equal to 156.3 µg/plate in the TA 100 strain, superior or equal to 312.5 µg/plate in the TA 1535 strain and superior or equal to 1250 µg/plate in the TA 98 strain.

No noteworthy toxicity was noted at any dose-levels in the TA 102 strain.

The test item did not induce any noteworthy increase in the number of revertants, in any of the five tested strains, in either experiment.

Experiments with S9 mix

Using the direct plate incorporation method, a moderate toxicity was only observed at dose-levels superior or equal to 2500 µg/plate in the TA 1537 strain.

Using the pre-incubation method, a moderate to strong toxicity was noted at dose-levels superior or equal to 625 µg/plate in the TA 1535, TA 1537, TA 98 and TA 100 strains, and superior or equal to 1250 µg/plate in the TA 102 strain.

The test item did not induce any noteworthy increase in the number of revertants in the TA 1537, TA 98, TA 100 and TA 102 strains in any experiments, or in the TA 1535 strain in the second experiment performed according to the pre-incubation method.

 

An increase in the number of revertants was noted at 5000 µg/plate in the TA 1535 strain in the first experiment (direct plate incorporation method). This increase exceeded the positive threshold of 3-fold the vehicle control value with an evidence of a dose-response relationship. Moreover, the corresponding mean number of revertants was above the vehicle control historical range.

Noteworthy increases in the number of revertants were then observed in the third experiment (under the same experimental conditions, i.e. direct plate incorporation method, and using a narrower range of dose-levels). These increases did not reach the positive threshold of 3-fold the vehicle control value, however there was an evidence of a dose-response relationship and the mean numbers of revertants at the four higher dose-levels (i.e. superior or equal to 12

50 µg/plate) were above the historical data range. Despite the positive threshold was not reached in the confirmatory experiment, the dose-related increases exceeding the historical data were reproducible, and they were thus considered to be biologically relevant.

Under the experimental conditions of this study, the test item showed a mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium TA 1535 strain in the presence of a rat liver metabolizing system, following the direct place incorporation method only.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
September - December 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
2015
Deviations:
no
GLP compliance:
yes
Type of assay:
other: in vitro gene mutation study in mammalian cells
Target gene:
hprt locus
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: Dr Donald Clive, Burroughs Wellcome Co.
- Storage at Covance: as frozen stocks in liquid notrogen.
Each batch of frozen cells was purged of mutants and confirmed to be mycoplasma free.
For each experiment, at least one vial was thawed rapidly, the cells diluted in RPMI 10 and incubated at 37+/-1°C. When the cells were growing well, subcutltures were established in an appropriate number of flasks.

MEDIA USED
- Type and identity of media: RPMI 1640 media containing L-glutamine and HEPES
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced rat liver post-mitochondrial fraction (S-9)
Test concentrations with justification for top dose:
The solubility limit in culture medium was approximately 67.31 to 134.6 µg/mL, as indicated by precipitation at the higher concentration which persisted for approximately 3 hours after test article addition. A maximum concentration of 500 µg/mL was selected for the cytotoxicity Range-Finder Experiment in order that treatments were performed up to a precipitating concentration. Concentrations selected for the Mutation Experiment were based on the results of this cytotoxicity Range-Finder Experiment.

Range finder (+/-S9): 1.563-3.125-6.25-12.5-25-50 mg/ml
Mutation experiment (-S9): 1-2-2.5-3-3.5-4-4.5-5-5.5-6-7.5 mg/ml
Mutation experiment (+S9): 2.5-5-7.5-10-12-14-16-18-20-22.5-25 mg/ml
Vehicle / solvent:
DMSO
Preliminary solubility data indicated that 3.3.5-trimethylcyclohexyl acrylate was miscible with anhydrous analytical grade dimethyl sulphoxide (DMSO) at concentrations up to approximately 215 mg/mL.
Test article stock solutions were prepared by formulating 3.3.5-trimethylcyclohexyl acrylate under subdued lighting in DMSO, with the aid of vortex mixing, to give the maximum required concentration. Subsequent dilutions were made using DMSO. The test article solutions were protected from light and used within approximately 2.5 hours of initial formulation.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
benzo(a)pyrene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium; in agar (plate incorporation); preincubation; in suspension; as impregnation on paper disk
- Cell density at seeding (if applicable):

DURATION
- Preincubation period: 3h
- Exposure duration: 7d
- Expression time (cells in growth medium): 7d

NUMBER OF CELLS EVALUATED: At the end of the expression period, cell concentrations in the selected cultures were determined using a Coulter counter and adjusted to give 1 x 105 cells/mL in readiness for plating for 6TG resistance.

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency
- Any supplementary information relevant to cytotoxicity: Cloning Efficiency (CE) in any given culture is therefore: CE = P/No of cells plated per well, and as an average of 1.6 cells/well were plated on all survival and viability plates, CE = P/1.6.
Percentage Relative Survival (% RS) in each test culture was determined by comparing plating efficiencies in test and control cultures thus: % RS = [CE (test)/CE (control)] x 100.
To take into account any loss of cells during the 3 hour treatment period, percentage relative survival values for each concentration of test article were adjusted as follows: Adjusted % RS = [% RS x Post-treatment cell concentration for test article treatment] / Post-treatment cell concentration for vehicle control


- OTHER: metabolic activation system
The mammalian liver post-mitochondrial fraction (S-9) used for metabolic activation was obtained from Molecular Toxicology Incorporated, USA where it is prepared from male Sprague Dawley rats induced with Aroclor 1254. The batches of S-9 were stored frozen in aliquots at <-50°C prior to use (Booth et al., 1980). Each batch was checked by the manufacturer for sterility, protein content, ability to convert known promutagens to bacterial mutagens and cytochrome P-450-catalyzed enzyme activities (alkoxyresorufin-O-dealkylase activities).
The S-9 mix was prepared in the following way: G6P (180 mg/mL), NADP (25 mg/mL), KCl (150 mM) and rat liver S-9 were mixed in the ratio 1:1:1:2. For all cultures treated in the presence of S-9, an aliquot of the mix was added to each cell culture to achieve the required final concentration of test article in a total of 20 mL. The final concentration of the liver homogenate in the test system was 2%.
Rationale for test conditions:
Acceptance Criteria: The assay was considered valid if the following criteria were met:
1. The MF in the concurrent negative control was considered acceptable for addition to the laboratory historical negative control database,
2. The MF in the concurrent positive controls induced responses that were compatible with those generated in the historical positive control database and give a clear, unequivocal increase in MF over the concurrent negative control,
3. The test was performed with and without metabolic activation,
4. Adequate numbers of cells and concentrations were analysable.
Evaluation criteria:
For valid data, the test article was considered to induce forward mutation at the hprt locus in mouse lymphoma L5178Y cells if:
1. The MF at one or more concentrations was significantly greater than that of the vehicle control (p=0.05)
2. There was a significant concentration-relationship as indicated by the linear trend analysis (p=0.05)
3. The results were outside the historical vehicle control range.
Results that only partially satisfied the assessment criteria described above were considered on a case-by-case basis. Positive responses seen only at high levels of cytotoxicity required careful interpretation when assessing their biological relevance. Extreme caution was exercised with positive results obtained at levels of RS lower than 10%.
Statistics:
Statistical significance of mutant frequencies was carried out according to the UKEMS guidelines (Robinson et al., 1990). The control log mutant frequency (LMF) was compared with the LMF from each treatment concentration and the data were checked for a linear trend in mutant frequency with test article treatment. These tests require the calculation of the heterogeneity factor to obtain a modified estimate of variance.
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
Toxicity
In the cytotoxicity Range-Finder Experiment, six concentrations were tested in the absence and presence of S-9 ranging from 15.63 to 500 µg/mL (limited by solubility of the test article formulation in culture medium). Upon addition of the test article to the cultures, precipitate was observed at the highest three concentrations (125 to 500 µg/mL) but no precipitate was observed following the 3 hour treatment incubation period. The highest concentrations to give >10% RS were 31.25 µg/mL in the absence of S-9 and 125 µg/mL in the presence of S-9, which gave 68% and 37% RS, respectively. No marked changes in osmolality or pH were observed in the Range-Finder Experiment at the highest concentration tested (500 µg/mL), compared to the concurrent vehicle controls (individual data not reported).
In the Mutation Experiment, eleven concentrations, ranging from 10 to 75 µg/mL in the absence of S-9 and from 25 to 250 µg/mL in the presence of S-9, were tested. Upon addition of the test article to the cultures, precipitate was observed at the highest seven concentrations in the presence of S-9 (120 to 500 µg/mL) but no precipitate was observed in the absence and presence of S-9 following the 3 hour treatment incubation period. Seven days after treatment, the highest two concentrations in the absence of S-9 (60 and 75 µg/mL) and the highest four concentrations in the presence of S-9 (180 to 250 µg/mL) were considered too toxic for selection to determine viability and 6TG resistance. All other concentrations were selected in the absence and presence of S-9. The highest concentrations analysed were 55 µg/mL in the absence of S-9 and 160 µg/mL in the presence of S-9, both of which gave 7% RS. Steep concentration related toxicity was observed under both treatment conditions. In the absence of S-9, cultures tested at 45, 50 and 55 µg/mL gave 30%, 7% and 7% RS, respectively. Furthermore at 50 µg/mL, the two replicate cultures gave RS values of 15% and 2%. All three concentrations were analysed. In the presence of S-9, cultures tested at 140 and 160 µg/mL gave 41% and 7% RS, respectively, therefore both concentrations were analysed.

Mutation
The acceptance criteria were met and the study was accepted as valid.
When tested up to highly toxic concentrations, no statistically significant increases in MF, compared to the vehicle control MF, were observed at any concentration analysed in the absence and presence of S-9 following treatment with 3.3.5-trimethylcyclohexyl acrylate and there were no statistically significant linear trends. An increase in MF was seen in one culture at 50 µg/mL in the absence of S-9, but this was observed in the culture giving 2% RS (which was considered excessively toxic).
In this study, steep concentration-related toxicity was observed over very narrow ranges (45 to 55 µg/mL in the absence of S-9 and 140 to 160 µg/mL in the presence of S-9) and the maximum concentrations analysed under both treatment conditions gave 7% RS, which is below the recommended upper toxicity limit of 10-20% RS. In the absence of S-9, cultures analysed at 50 and 55 µg/mL both gave mean RS values of 7%: At 50 µg/mL the individual RS values were 15% and 2% and at 55 µg/mL they were 6% and 8%. No increases in MF were seen apart from the single replicate culture at 50 µg/mL which gave 2% RS. Similarly in the presence of S-9, cultures analysed at 160 µg/mL gave a mean RS value of 7%, with individual values of 6% and 8% and no marked increases in MF were seen in either replicate culture.
Thus, the only elevated MF value was observed in a single culture which gave 2% RS and no such increases in MF were observed in any culture giving >6% RS in the absence or presence of S-9, therefore 3.3.5-trimethylcyclohexyl acrylate was considered not mutagenic in this test system.
Conclusions:
It is concluded that 3.3.5-trimethylcyclohexyl acrylate did not induce mutation at the hprt locus of L5178Y mouse lymphoma cells when tested up to highly toxic concentrations for 3 hours in the absence and presence of a rat liver metabolic activation system (S-9) under the experimental conditions described.
Executive summary:

3.3.5-trimethylcyclohexyl acrylate was assayed for the ability to induce mutation at the hypoxanthine-guanine phosphoribosyl transferase (hprt) locus (6-thioguanine [6TG] resistance) in mouse lymphoma cells using a fluctuation protocol. The study consisted of a cytotoxicity Range-Finder Experiment followed by one Mutation Experiment, conducted in the absence and presence of metabolic activation by an Aroclor 1254-induced rat liver post-mitochondrial fraction (S-9). The test article was formulated in anhydrous analytical grade dimethyl sulphoxide (DMSO). A 3 hour treatment incubation period was used for all experiments.

In the cytotoxicity Range-Finder Experiment, six concentrations were tested in the absence and presence of S-9, ranging from 15.63 to 500 µg/mL (limited by solubility of the test article formulation in culture medium). The highest concentrations to give >10% relative survival (RS) were 31.25 µg/mL in the absence of S-9 and 125 µg/mL in the presence of S-9, which gave 68% and 37% RS, respectively.

In the Mutation Experiment, eleven concentrations, ranging from 10 to 75 µg/mL in the absence of S-9 and from 25 to 250 µg/mL in the presence of S-9, were tested. Seven days after treatment, the highest concentrations analysed to determine viability and 6TG resistance were 55 µg/mL in the absence of S-9 and 160 µg/mL in the presence of S-9, both of which gave 7% RS. Steep concentration-related toxicity was observed under both treatment conditions. In the absence of S-9, cultures tested at 45, 50 and 55 µg/mL gave 30%, 7% and 7% relative survival (RS), respectively. Furthermore at 50 µg/mL, the two replicate cultures gave RS values of 15% and 2%. All three concentrations were analysed. In the presence of S-9, cultures tested at 140 and 160 µg/mL gave 41% and 7% RS, respectively, therefore both concentrations were analysed.

Vehicle and positive control treatments were included in the Mutation Experiment in the absence and presence of S-9. Mutant frequencies (MF) in vehicle control cultures fell within acceptable ranges and clear increases in mutation were induced by the positive control chemicals 4-nitroquinoline 1-oxide (NQO) (without S-9) and benzo(a)pyrene (B[a]P) (with S-9). Therefore the study was accepted as valid.

When tested up to highly toxic concentrations, no statistically significant increases in MF, compared to the vehicle control MF, were observed at any concentration analysed in the absence and presence of S-9 and there were no statistically significant linear trends. An increase in MF was seen in one culture at 50 µg/mL (giving 2% RS) in the absence of S-9, but toxicity was considered excessive in this culture. No marked changes in MF were seen in both replicate cultures at 55 µg/mL (which gave 6% and 8% RS), therefore the isolated observation in the single, excessively toxic culture at 50 µg/mL was considered not biologically relevant.

It is concluded that 3.3.5-trimethylcyclohexyl acrylate did not induce mutation at the hprt locus of L5178Y mouse lymphoma cells when tested up to highly toxic concentrations for 3 hours in the absence and presence of a rat liver metabolic activation system (S-9) under the experimental conditions described.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Mode of Action Analysis / Human Relevance Framework

In vitro gene mutation study in bacteria (Chevallier 2016a)

The objective of this study was to evaluate the potential of the test item to induce reverse mutations in Salmonella typhimurium (OECD 471).

Treatments were performed according to the direct plate incorporation method except for the second experiment with S9 mix, which was performed according to the pre-incubation method (60 minutes, 37°C). Five strains of bacteria Salmonella typhimurium were used: TA 1535, TA 1537, TA 98, TA 100 and TA 102. Each strain was exposed to at least five dose-levels of the test item (three plates/dose-level). After 48 to 72 hours of incubation at 37°C, the revertant colonies were scored. The evaluation of the toxicity was performed on the basis of the observation of the decrease in the number of revertant colonies and/or a thinning of the bacterial lawn.In experiments without S9 mix : the test item did not induce any noteworthy increase in the number of revertants, in any of the five tested strains, in either experiment.

In experiments with S9 mix, the test item did not induce any noteworthy increase in the number of revertants in the TA 1537, TA 98, TA 100 and TA 102 strains in any experiments, or in the TA 1535 strain in the second experiment performed according to the pre-incubation method. However an increase in the number of revertants was noted at 5000 µg/plate in the TA 1535 strain in the first experiment (direct plate incorporation method). This increase exceeded the positive threshold of 3-fold the vehicle control value with an evidence of a dose-response relationship. Moreover, the corresponding mean number of revertants was above the vehicle control historical range.

Noteworthy increases in the number of revertants were then observed in the third experiment (under the same experimental conditions, i.e. direct plate incorporation method, and using a narrower range of dose-levels). These increases did not reach the positive threshold of 3-fold the vehicle control value, however there was an evidence of a dose-response relationship and the mean numbers of revertants at the four higher dose-levels (i.e. superior or equal to 50 µg/plate) were above the historical data range. Despite the positive threshold was not reached in the confirmatory experiment, the dose-related increases exceeding the historical data were reproducible, and they were thus considered to be biologically relevant.

Under the experimental conditions of this study, the test item showed a mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium TA 1535 strain in the presence of a rat liver metabolizing system, following the direct place incorporation method only.

 

In vitro cytogenicity / micronucleus study (Chevallier 2016b):

The objective of this study (OECD 487) was to evaluate the potential of the test item, to induce an increase in the frequency of micronucleated cellsin the mouse cell line L5178Y TK+/-.

Following the 3- and 24-hour treatments without S9-mix, a slight to severe cytotoxicity was induced at dose-levels = 0.01 mM, as shown by a 26 to 100% decrease in the PD. Neither statistically significant nor dose-related increase in the frequency of micronucleated cells was noted at any of the tested dose-levels relative to the vehicle controls. None of the analyzed dose-levels showed frequency of micronucleated cells of each replicate culture above the vehicle control historical range. These results met the criteria of a negative response.

 In the experiments with S9 mix, neither statistically significant nor dose-related increase in the frequency of micronucleated cells was noted at any of the tested dose-levels relative to the vehicle control. None of the analyzed dose-levels showed frequency of micronucleated cells of each replicate culture above the vehicle control historical range. These results met the criteria of a negative response.

Under the experimental conditions of the study, 3.3.5-trimethylcyclohexyl acrylate, the test item did not induce any chromosome damage, or damage to the cell division apparatus, in cultured mammalian somatic cells, using L5178Y TK+/- mouse lymphoma cells, either in the presence or absence of a rat liver metabolizing system.

 

In vitro gene mutation study in mammalian cells (Lloyd 2017):

3.3.5-trimethylcyclohexyl acrylate was assayed for the ability to induce mutation at the hypoxanthine-guanine phosphoribosyl transferase (hprt) locus (6-thioguanine [6TG] resistance) in mouse lymphoma cells using a fluctuation protocol (OECD 476). The study consisted of a cytotoxicity Range-Finder Experiment followed by one Mutation Experiment, conducted in the absence and presence of metabolic activation by an Aroclor 1254-induced rat liver post-mitochondrial fraction (S-9). The test article was formulated in anhydrous analytical grade dimethyl sulphoxide (DMSO). A 3 hour treatment incubation period was used for all experiments.

When tested up to highly toxic concentrations, no statistically significant increases in MF, compared to the vehicle control MF, were observed at any concentration analysed in the absence and presence of S-9 and there were no statistically significant linear trends. An increase in MF was seen in one culture at 50 µg/mL (giving 2% RS) in the absence of S-9, but toxicity was considered excessive in this culture. No marked changes in MF were seen in both replicate cultures at 55 µg/mL (which gave 6% and 8% RS), therefore the isolated observation in the single, excessively toxic culture at 50 µg/mL was considered not biologically relevant.

It is concluded that 3.3.5-trimethylcyclohexyl acrylate did not induce mutation at the hprt locus of L5178Y mouse lymphoma cells when tested up to highly toxic concentrations for 3 hours in the absence and presence of a rat liver metabolic activation system (S-9) under the experimental conditions described.

Additional information

Justification for classification or non-classification

Based on the weight of evidence, 3,3,5 -trimethylcyclohexyl acrylate is considered to be not mutagenic. No classification is required according to the Regulation EC n°1272/2008.